【摘要】 目的纯化重组细菌Pw鄄1/pRSETB/BL21的表达产物肺吸虫重组抗原,复性后以ELISA法检测肺吸虫病患者、其他寄生虫病患者和正常人血清,评价其敏感性、特异性和应用前景。方法通过分步洗涤及透析对细菌重组蛋白进行初步纯化,进一步做快速液相层析纯化,比较前后两法的纯化效果。初步纯化产物经氧化/还原型谷胱苷肽复性后即为重组蛋白抗原(以下简称重组抗原)。用该重组抗原,以ELISA法检测肺吸虫、血吸虫、肝吸虫、囊虫和包虫病患者以及正常人血清中相应抗体,并与卫氏肺吸虫粗抗原做比较。结果经分步洗涤及透析纯化后的重组抗原纯度已较高,快速液相层析进一步纯化效果不明显。以初步纯化后经复性的重组抗原进行ELISA法检测肺吸虫病患者血清,敏感性为91.07%,与其他寄生虫病患者和正常人血清均无交叉反应;粗抗原检测肺吸虫病患者血清敏感性虽为100%,但与其他寄生虫病患者和正常人血清交叉反应率分别为血吸虫病11.43%,肝吸虫病4.84%,囊虫病3.22%,包虫病5.88%,正常人为2.5%。结论研究制备的重组抗原应用于肺吸虫病的免疫诊断特异性较高。更多还原

【Abstract】 Objective: To purify recombinant Paragonimus Westermani(Pw) adult antigen expressed in bactieria Pw-1/pRSET B/BL 21. After renature, the purified products were applied to detect the sera collected from paragonimiasis patients, other parasitosis and normal persons with ELISA and the specificity, sensitivity and application prospect of ELISA using Pw were evaluated. Methods: After primary purification of the recombinent protein by dialyse, the primary purified protein was further purified by Fast Protein Liquid Chromatography(FPLC), then the effects of the two steps of purification methods were compared. The primary purified products by Oxidation/reduction GSH renatured were to abtain the recombinant protein antigen. Finally, the sera collected from patients of paragonimiasis, schistosomiasis, clonorchiasis sinensis, cysticerciasis, echinococcosis and normal persons were detected for corresponding antibodies against the recombinant protein antigen and the Pw adult crude antigen respectively by ELISA. Results: FPLC, the sensitivity of the recombinant antigen tested by paragonimiasis patients’sera was 91.07%, and there was not any cross reaction with the sera collected from other parasitosis and normal persons. In contrast, the sensitivity of crude antigen was 100%, while the percentages of cross reaction were as follows: schistosomiasis 11.43%, clonorchiasis sinensis 4.84%, cysticerciasis 3.22%, echinococcosis 5.88%, normal person 2.5%. Conclusion: The primary purified antigen applied in the immunodiagnosis has higher specificity.更多还原